NMR studies of an oligonucleotide with an unusual structure induced by platinum anti-cancer drugs.

The 31P NMR spectra of Pt(en)[d(T1A2T3G4G5G6T7A8C9C10C11A12T13A14)] (14-mer) and Pt(en)[d(A2T3G4G5G6T7A8C9C10C11A12T13)] (12-mer) (en = ethylenediamine) each contain two signals far downfield (ca. -2.9 and -2.6 ppm from trimethylphosphate standard), two signals slightly downfield, and at least one signal slightly upfield of the normal range (ca -4.0 to -4.4 ppm). This pattern suggested a distorted structure. The unusual 31P signals of the 12-mer were assigned by analogy to signals of the 14-mer previously assigned by 17O-labeling methods. A combination of heteronuclear multiple-quantum coherence, one-dimensional and two-dimensional nuclear Overhauser effect (1D and 2D-NOE) and homonuclear shift correlation spectroscopy (COSY) experiments assigned all aromatic 1H signals of the 12-mer except H8 of G5 or G6. One of these H8 signals is missing from the spectrum and the nucleotide is labeled Gm. The other H8 is the most downfield signal and has a strong NOE to its H1'. Since this strong NOE indicates that this nucleoside exists in a syn conformation, it is labeled Gs. A strong NOE was observed between the Gs and A8 H8 signals. Several lines of evidence suggested a hairpin-like structure with a loop region (G6T7A8C9) and a stem region involving A2T3G4G5 and C10C11A12T13. The 31P signals for the stem region are within or slightly outside the normal range. 3JH3'-P values (3-6 Hz), measured by a 2D-J experiment, of stem nucleotides were characteristic for a DNA duplex. Imino signals for base pairs A2T13, T3A12, G4C11, and probably G5C10, and the observation of internucleotide NOE connectivities for these nucleotides (e.g. between an H8 signal and the H1' signal of the 5' nucleotide) suggested a right-handed helical structure. For the loop region, a distorted sugar-phosphate backbone is indicated by far downfield positions of the G5pG6 and A8pC9 31P signals, the 3JH3'-P values for C9p (8.0 Hz) and A8p (6.8 Hz), and the absence of H3'-P coupling for G5p. In the loop region, no imino signals or internucleotide NOEs characteristic of a right-handed duplex were observed. However, A8H8, C9H6, and C10H6 each exhibited unusual internucleotide NOEs to the H4' signal of the 5' residue. NOE crosspeaks between T7 1H signals and signals attributed to sugars of the Gs and Gm suggested that the T7 moiety is located within the space encircled by the loop. The few NOE crosspeaks, pH dependence, and Cu2+ broadening of C9 1H signals indicate an isolated location accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)